Development of a cofactor-dependent enzymatic ligation-triggered rolling circle amplification-assisted CRISPR-Cas12a strategy for ATP detection via a personal glucometer in meat freshness evaluation

Adenosine triphosphate (ATP), a central molecule in microbial energy metabolism, serves as a critical indicator for assessing food freshness. Herein, we developed a portable and sensitive ATP quantitative platform by integrating a cofactor-dependent enzymatic ligation-triggered rolling circle amplification (RCA)-assisted CRISPRCas12a strategy with a glucometer readout. T4 DNA ligase recognizes ATP to form circular DNA templates, initiating RCA reactions that generate amplicons. These amplicons activate CRISPR-Cas12a to cleave ssDNAinvertase conjugates, releasing invertase. The released invertase hydrolyzes sucrose into glucose, which is quantified using a glucometer. Glucometer signals correlate linearly with ATP concentrations from 5 to 200 nmol/L, with a detection limit of 1.08 nmol/L. Furthermore, the linear relationship between ATP levels on meat surfaces and total viable counts was validated. This strategy's accuracy was confirmed through fluorescence recovery experiments and commercial ATP kits, yielding consistent results that highlight the practical application potential of this method for assessing meat freshness.